Compounds for determining the activity of phospholipase A2

ABSTRACT

Compounds for determining the activity of phospholipase A 2 , are described herein, and include embodiments having formula (1) 
     
       
         
         
             
             
         
       
         
         
           
             wherein 
             L 1  is derived from an ether (R 1 —OR 2 ) m , wherein R 1  and R 2  are independently selected and are derived from a hydrocarbon having 1 to 12 carbon atoms, with m being an integer from 1 to 4, or from a hydrocarbon R having 1 to 20 carbon atoms; 
             F is unsubstituted or substituted pyrene as a flouraphore; 
             Q is a quencher, and 
             L 2  is C(O)-L 1  or C(O)-L 1 -NH, wherein L 1  is as defined above. These compounds may be used to determine the activity of phospholipase A 2 , in particular PAF-AH.

RELATED APPLICATION

This application claims priority to PCT Application No. PCT/EP02/11852filed Oct. 23, 2002 and European Application No. 01129137.4 filed Dec.7, 2001.

FIELD OF INVENTION

The invention relates to compounds which are suitable to determine theactivity of phospholipase A₂ (PLA₂), in particular ofPlatelet-Activating Factor Acetylhydrolase (PAF-AH).

BACKGROUND

The Platelet-Activating Factor (PAF) is a biologically activephospholipid involved in physiological and pathophysiological processes.This factor is converted to biologically inactive products by a specificacetyl hydrolase, i.e. Platelet-Activating Factor Acetylhydrolase(PAF-AH, EC 3.1.1.47, also referred to as lipoprotein-associatedphospholipase A₂), which cleaves off the acetate group at the sn-2position. In addition to PAF, the substrate specificity also comprisesoxidized phospholipids having polar substituents at the ω-position ofthe sn-2 ester. These altered substances are often found in oxidizedlipoproteins. Since oxidized lipoproteins, in addition to PAF andoxidized phospholipids, also play a role in a great variety of diseases,it may be assumed that the enzyme also has a decisive influence on thegenesis of said diseases.

SUMMARY OF THE INVENTION

In recent years, PAF-AH has become more and more important in research.In particular, abnormalities in PAF-AH activity have been found in agreat variety of diseases. Series of clinical tests and the beginning ofexaminations of the enzyme, prepared in a recombinant manner, as atherapeutic agent (phase III of the clinical experiments will begin inearly 2001 in the indicated field of sepsis (www.icos.com)) are showingits great importance for humans. Therefore, it is required to have awell-feasible method for determining the activity of PLA₂ in generaland, in particular, of PAF-AH.

In order to determine the activity of PAF-AH, the literature describes agreat variety of methods, such as radioactivity measurements, UVmeasurements or fluorescence measurements. In nearly all of thesemethods, the actual reaction of the substrate with a sample containingthe enzyme is followed by one or more process step(s) (e.g. extraction,consequent reactions to indicators), so that continuous measurement ofthe enzyme kinetics is not possible.

The oldest and currently most widely used methods of determination arebased on radioactivity measurements (M. E. Blank et al., J. Biol. Chem,1981, 256, 175-178). These involve reacting a [³H]-acetate-markednatural substrate with the sample to be investigated, wherebyradioactively labeled acetic acid is cleaved off. After chromatographicprocessing of the test preparation, the cleaved-off acetic acid can beradioactively determined. However, chromatographic processing istime-consuming and, in part, also incurs high costs.

H. S. Hendrickson et al., Anal. Biochem., (1999), 276, 27-35 discloseintramolecularly quenched BODIPY-labeled phospholipid analogs in methodsfor determining PLA₂ and PAF-AH. One of said analogs is1-(N-(BODIPY-FL-pentanoyl)-11-aminoundecyl)-2-((N-(2,4-dinitrophenyl)amino)-8-amino-octanoyl)-sn-glycero-3-phophocholine.BODIPY serves as a fluorophore.

However, this compound has quite severe drawbacks. Thus, the use of theaforementioned compound for determining the activity of PLA₂, inparticular of PAF-AH, does not allow any statements as to changes of thelateral diffusion rate in membranes. Moreover, the properties of theaforementioned compound differ greatly from the properties of naturalphospholipids.

Therefore, it is an object of the present invention to provide a meansallowing to determine the activity of PLA₂, in particular of PAF-AH,without the disadvantages known from the prior art.

DETAIL DESCRIPTION OF THE PREFERRED EMBODIMENTS

According to the invention, this is achieved by a compound of formula(1)

whereinL₁ is derived from an ether (R¹—OR²)_(m), wherein R¹ and R² areindependently selected and are derived from a hydrocarbon having 1 to 12carbon atoms, with m being an integer from 1 to 4, or from a hydrocarbonR having 1 to 20 carbon atoms;F is unsubstituted or substituted pyrene as a fluorophore;Q is a quencher, andL₂ is C(O)-L₁ or C(O)-L₁-NH, wherein L₁ is as defined above.

Although the compounds of the invention comprise unsubstituted orsubstituted pyrene as a fluorophore, they are not fluorescent, becausesuch fluorescence is quenched by the quencher which is also contained inthe compound of the invention. Under the influence of PLA₂, inparticular PAF-AH, the quencher is cleaved off and spatially separatedfrom the remaining compound. This deactivates the quenching offluorescence, so that an increase in fluorescence is observed. Thisincrease in the intensity of fluorescence is generally directlyproportional to the activity of PLA₂ and is thus an indicator for theactivity of PLA₂, in particular of PAF-AH.

Surprisingly, it has been found that the use of the aforementionedcompound for determining the activity of PLA₂, in particular of PAF-AH,allows statements to be made as to changes of the lateral diffusion ratein membranes. Moreover, the properties of the aforementioned compounddiffer greatly from the properties of natural phospholipids. Further,the compounds of the invention allow a determination of activity to becarried out in a particularly effective and easy manner. Activity can bemeasured in a direct and continuous manner.

As outlined above, unsubstituted or substituted pyrene is present as afluorophore in the compound of the invention. Said fluorophore may bebound, via its 1-position, to L₁. If said pyrene is substituted, use ismade of substituents which do not have a substantial influence on itsproperties as a fluorophore. Such substituents are known to the personskilled in the art. They may be amino groups or carbonyl groups orheteroatoms, such as N, S, or P, which substitute the C atoms of pyrene.The compound of the invention may comprise one or more independentlyselected substituents.

Compared with other fluorophores, such as NBD(N-(7-nitrobenz-2-oxa-1,3-diazole-4-yl)) or BODIPY(4,4-difluoro-4-bora-3a,4a-diaza-s-indacene), the above pyrenes have theadvantages of a lower polarity, a great distance between the extinctionwavelength and the emission wavelength, the ability to work in aqueoussolution, a favourable distribution in cells, the ability to beincorporated in membranes without quenching of fluorescence as well as apH-independent fluorescence. A further positive property of pyrenes isthe formation of excimers at higher concentrations and a simultaneousshift of the emission wavelength to greater wavelengths. The formationof excimers allows an assessment of the concentration of thefluorophore.

According to the above formula (1), the compounds of the inventioncomprise a quencher. Quenchers are compounds known per se in the fieldof fluorescence, which reduce or extinguish the fluorescence intensityof the fluorophore. Examples of such quenchers include phenyl residues,which are substituted with one or more, in particular two, nitro groups,such as e.g. the 2,4-dinitrophenyl residue. These quenchers enableparticularly efficient intramolecular reduction or quenching of thefluorescence intensity of the fluorophores.

In the compounds of the invention, the fluorophore is bound to the Oatom at the sn 1 position of the glycerol via a linker. The quencher isalso bound to the glycerol via a linker, namely at the sn-2 position ofthe glycerol. In doing so, the linkers are selected such, with regard totheir dimensions, in particular their length, that optimal quenching ofthe fluorescence intensity can be observed. The linker L₂, by which thequencher is bound to the glycerol residue, is hydrolysable due to theO—C(O) group by PLA₂, in particular by PAF-AH. The quencher is cleavedoff from the compound of the invention, together with the linker, byhydrolysis; the fluorescence intensity then increases, becauseintramolecular quenching is no longer possible. In contrast thereto, thelinker L1, by which the fluorophore is bound to the glycerol, must notbe cleaved off by PLA₂, in particular not by PAF-AH. Rather, the linkerneeds to be metabolically stable to detect the fluorescence intensity ofthe linkers.

As already outlined above, the linker may be derived from an ether(R¹—OR²)_(m), wherein R¹ and R² are independently selected and arederived from a hydrocarbon having 1 to 12 carbon atoms, with m being aninteger from 1 to 4.

The term “hydrocarbons” relates to organic compounds consistingexclusively of carbon and hydrogen. Examples include alkanes, alkenes,and alkynes. Alkenes and alkynes may include several unsaturated bonds.According to the above formula (1), two H atoms of the aforementionedhydrocarbons are substituted so that they can perform their function asor in the linker. Said substitution may be effected, in particular, atterminal C atoms of the hydrocarbons.

In a preferred embodiment of the compound of the invention, R¹ and R²are (CH₂)_(n), wherein n is an integer from 2 to 12. In particular, L₁represents (CH₂)₄—O—(CH₂)₁₀. Linkers comprising CH₂ chains areespecially favourable because they have particular metabolic stability,which ensures that the fluorophore remains in the compound of theinvention, despite enzymatic treatment, and can be excited to fluoresce.

As outlined above, the linker L₁ may be derived from a hydrocarbon R. Ris preferably (CH₂)_(o), wherein o is an integer from 1 to 20, inparticular 2 to 6. It has been found that linkers of this kind areparticularly favourable, because they are metabolically stable, thuspreventing any undesired cleavage of the fluorophore from the compoundof the invention during treatment with enzymes.

The compound of the invention comprises a second linker L₂, by which thequencher is bound to the sn-2 position of the glycerol. As outlinedabove, L₂ is C(O)-L, or C(O)-L₁-NH, wherein L₁ is as defined above.

In a preferred embodiment, L₂ is C(O)—(CH₂)_(p) or C(O)—(CH₂)_(p)—NH,wherein p is an integer from 1 to 20, in particular 2 to 6, and, inparticular, L2 means C(O)—(CH₂)₅—NH. These linkers are particularlywell-suited because, on the on hand, the CH2 chain is metabolicallystable, thus avoiding undesired cleavage of the quencher at thislocation, and, on the other hand, the ester group —C(O)—O— of PLA₂, inparticular of PAF-AH, is purposefully and selectively cleaved by theseenzymes.

One advantage of pyrene binding to the 1-position of glycerol is that itis metabolically stable there; no disruptions of the test byphospholipase A1 activities, which may also be present in the sample tobe investigated, are observed.

The phosphatidylcholine residue is present at the sn 3-position ofglycerol. This favourably ensures that the compounds of the inventionexhibit the biophysical behaviour of natural phospholipids.

In a particularly preferred embodiment, the compound of the inventionhas a structure of formula (2)

This compound of the invention according to formula (2) enablesmeasurement of the activity of PLA₂, in particular of PAF-AH, in aparticularly easy and favourable manner.

In order to prepare the compound of the invention, the skilled personcan resort to conventional reactions and methods in synthetic organicchemistry. For example, the fluorophore may first be bound to a linker,then the second end of the linker can be introduced at the sn-1 positionof the glycerol. The phosphocholine residue may then be introduced atthe sn-3 position of the glycerol, wherein the OH group at the sn-2position of the glycerol may be protected by a protective group, forexample by a benzoyl group. The protective group may be subsequentlycleaved off in a conventional manner, and then the second linker may beinserted, optionally together with the quencher. The method of preparingthe compounds of the invention was described above without a detaileddescription of intermediate steps. For example, insertion of the linkerinto the fluorophore may be effected in several steps, such as thosedescribed, e.g., in the preparation example below.

The presence of the quencher in the compound of the invention causesintramolecular quenching of fluorescence. By cleaving off the quencherfrom the compound of the invention, intramolecular quenching is nolonger possible, so that an increase in the fluorescence intensity ofthe fluorophore is observed. Such cleavage of the quencher from thecompound of the invention may be effected by means of PLA₂, inparticular PAF-AH, namely by hydrolysis of the ester group at the sn-2position. The compounds of the invention are, therefore, particularlywell-suited to determine the activity of PLA₂, in particular of PAF-AH.

In such a method of determining the activity of PLA₂, in particular ofPAF-AH, a sample containing said enzyme is incubated with the compoundof the invention and fluorescence is measured in a conventional manner.In doing so, incubation is favourably effected under physiologicconditions, for example at pH 7.4 in an aqueous medium, e.g. buffer, andat 37° C., so as to enable measurement of the activity of the enzyme inan environment which is as natural as possible. The measurement offluorescence, i.e. the increase in fluorescence, may be effected in acontinuous manner. Such continuous measurement allows fluorescence to bepermanently determined over a longer period of time from a few minutesto several hours, in particular from 5 minutes to 0.5 hours.

The compounds of the invention are, therefore, particularly well-suitedto determine the enzymes PLA₂, in particular PAF-AH. These compoundsenable particularly effective, direct, easy, quick and low-costmeasurement. Furthermore, said activity measurement can be carried outin a continuous manner, i.e. over a longer period of time, thus allowingto obtain valuable information in relation to enzyme kinetics. Moreover,using the compounds of the invention very exact values are obtained,which allow the reliable statements to be made, in particular fortherapeutic and diagnostic purposes.

The activity measurement of enzymes may have a large number ofapplications, for example

-   -   as a diagnostic agent for differential diagnosis of a great        variety of biological samples in humans and animals (nose        secretion, sperm, lung secretion (BAL), blood or its        constituents, isolated cell populations, tissue biopsies, gall        secretion, intestinal secretion, stool specimens, such as    -   to detect a great variety of metabolic functions, e.g.        unspecific conditions, such as hyperlipidemia, conditions of        increased oxidative stress,    -   for the diagnose of acute inflammatory diseases, such as sepsis,        ARDS (lung failure), as well as inflammations of solitary organs        as well as, in particular, also of wound-healing disorders;    -   to detect chronic inflammatory conditions, such as rheumatoid        arthritis, and further diseases of the rheumatic type, Colitis        ulzerosa, Morb. Crohn;    -   in other local and systemic inflammatory conditions;    -   in allergy—and/or immunology-mediated diseases, e.g. allergies        to pollen and to latex, glomerulonephritis;    -   in diseases of the toxic genesis, e.g. toxic lung failure or        poisoning conditions;    -   in coronary diseases, e.g. acute coronary syndrom, angina        pectoris, Prinzmetal's angina, cardiac infarction, as well as        arteriosclerosis, acute cerebral insult;    -   in other conditions, such as after transplantations, operations,        other invasive procedures;    -   in connection with medical check-ups for the above-mentioned        medical syndromes;    -   for therapy monitoring of medical measures, such as    -   any kind of medicinal intervention, such as therapies using        recombinant enzymes, as well as all types of therapeutic        intervention involving changes in the activity of the        corresponding enzymes; in particular, in the above-mentioned        fields of therapeutic indication;    -   invasive measures, such as operations for removal of        inflammatory causes, operations including replacement or        mechanical support of the heart-lung function, operations on the        liver and also a great variety of transplantations; in        particular, in the above-mentioned fields of therapeutic        indication;    -   the combination of both.

Determining the activity of PLA₂, in particular of PAF-AH, using thecompounds of the invention, has a great number of advantages. Thus, theactivity of the enzyme can be determined without burdensome processsteps and without side reactions. The addition of suitable inhibitorsenables selective measurement of enzymes cleaving phospholipid esters inmixtures of various hydrolases. The determination of enzyme activityusing the compounds of the invention may be effected in the wells ofmicrotiter plates. Further, this determination of activity is suitablefor HTS. The continuous measurement enables detection of kinetic data,such as IC50 and KI values.

The following examples explain the present invention without limiting itthereto, however, with reference to FIGS. 1 and 2, wherein

FIG. 1 shows the typical time course of the increase in fluorescence dueto the effect of PAF-AH, and

FIG. 2 shows the relationship between different amounts of enzyme ordifferent dilutions of human sample material and the increase influorescence.

EXAMPLE 1 Preparation of1-O-{(10-[4-(pyrene-1-yl)-butoxy]-decyl}-2-(6-(2,4-dinitrophenyl)-aminohexoyl-rac-glycero-3-phosphocholine According to the invention

Dichloro-phosphoric acid-(2-bromoethylester)

To a solution of 42.5 g (0.275 mol) of phosphoryl chloride in 50 ml ofdichloromethane, 27 g (0.215 mol) of bromoethanol in 15 ml ofdichloromethane were added dropwise within 15 min at room temperature.After 15 min, a slight argon stream was passed through the reactionsolution for 18 hours. Destillation in an oil pump vacuum (73-74° C.,3*10⁻² mbar) yielded 50.5 g (76%) of a colorless liquid.

M (C₂H₄ ⁷⁸Br³⁵Cl₂O₂ ³¹P)=239 g/mol

EI-MS: m/z (%)=243 (0.5) [M], 241 (0.9) [M], 239 (0.6) [M], 163 (25),161 (41), 149 (18), 147 (30), 137 (36), 135 (55), 119 (23), 117 (34),109 (64), 108 (100), 107 (69), 106 (100), 99 (18), 47 (27). ¹H-NMR(CDCl₃): δ (ppm)=4.59 (2H, m, CH₂Br, ³J_(HH)=6.26 Hz, ³J_(HP)=10.34 Hz),3.13 (2H, m, CH₂O, ³J_(HH)=6.26 HZ, ⁴J_(HP)=1.05 Hz). ¹³C-NMR (CDCl₃): δ(ppm)=69.88 (1 CH₂O, ²J_(CP)=8.39 Hz), 27.32 (1 CH₂Br, ³J_(CP)=10.29Hz). IR (Film): {tilde over (ν)} (cm⁻¹)=3033w, 2971w, 2883w, 1453m,1423m, 1385w, 1306s, 1233m, 1180m, 1071s, 1016s, 963m, 908m, 845w, 759m.

4oxo-4-(pyrene-1-yl)-butyric acid

To a solution of 0.5 g (5 mmol) of succinic anhydride in 60 ml ofnitrobenzene, 1.32 g (5 mmol) of anhydrous AlCl₃ and 1 g (5 mmol) ofpyrene were added at 0° C. After a reaction time of 18 hours at roomtemperature, the solution was poured onto 30 ml of iced hydrochloricacid (25%), forming a precipitate. Filtration and recrystallization fromEtOH yielded 1.233 g (82%) of 4-oxo-4-(pyrene-1-yl)-butyric acid.

M (C₂₀H₁₄O₃)=302 g/mol

EI-MS: m/z (%)=303 (10) [MH⁺], 302 (42) [M], 230 (16), 229 (100), 202(11), 201 (65), 200 (24). HR-EI-MS (C₂₀H₁₄O₃) calc.: 302.0943; found:302.0943. ¹H-NMR ([D₆]-DMSO): δ (ppm)=8.81-8.12 (9H, m, CH_(arom)), 3.52(2H, t, COCH₂, ³J_(HH)=6.22 Hz), 2.82 (2H, t, CH₂CO₂H, ³J_(HH)=6.22 Hz).¹³C-NMR ([D₆]-DMSO): δ (ppm)=203.05 (1 CO), 173.87 (1 CO₂H),132.97-123.41 (9 CH_(arom), 7 C_(arom)), 36.87 (1 COCH₂), 28.52 (1CH₂CO₂H). IR (KBr): {tilde over (ν)} (cm⁻¹)=3426m, 3039m, 2984m, 2929m,2677w, 2594w, 1695s, 1665s, 1507w, 1404w, 1212m, 1123w, 1075w, 842m,713w.

4-(pyrene-1-yl)-butyric acid

1 g (2.78 mmol) of 4-oxo-4-(pyrene-1-yl)-butyric acid was dissolved in30 ml diethylene glycol and 0.5 9 (10 mmol) of hydrazine hydrate as wellas 0.56 g (10 mmol) of KOH added thereto. The reaction solution washeated under reflux for 2 hours and then poured onto iced hydrochloricacid (25%), forming a yellow precipitate. Filtration of the solid andrecrystallization from EtOH yielded 570 mg (71%) of4-(pyrene-1-yl)-butyric acid.

M (C₂₀H₁₆O₂)=288 g/mol

EI-MS: m/z (%)=289 (16) [MH⁺], 288 (62) [M], 216 (20), 215 (100), 213(11). HR-EI-MS C₂₀H₁₆O₂): calc.: 288.1150; found: 288.1150. ¹H-NMR([D₆]-DMSO): δ (ppm)=8.50-7.92 (9H, m, CH_(arom)), 3.36 (2H, m,CH₂C₁₆H₉), 2.41 (2H, m, CH₂CO₂H), 2.04 (2H, m, CH₂). ¹H-NMR (CDCl₃): δ(ppm)=8.32-7.86 (9H, m, CH_(arom)), 3.42 (2H, t, CH₂C₁₆H₉, ³J_(HH)=7.57Hz), 2.51 (2H, t, CH₂CO₂H, ³J_(HH)=7.00 Hz), 2.23 (2H, m, CH₂,³J_(HH)=7.57 Hz, ³J_(HH)=7.00 Hz). ¹³C-NMR ([D₆]-DMSO): δ(ppm)=174.74 (1CO), 136.39-123.39 (9 CH_(arom), 7 C_(arom)), 33.64-26.98 (1 CH₂CO₂H, 2CH₂). ¹³C-NMR (CDCl₃): δ (ppm)=177.48 (1 CO), 135.25-123.04 (9CH_(arom), 7 C_(arom)), 33.21-26.46 (1 CH₂CO₂H, 2 CH₂). IR (KBr): {tildeover (ν)} (cm⁻¹)=3447w, 3037w, 2950m, 2934w, 2874w, 1695s, 1431w, 1275m,1206m, 918w, 846s, 711w.

4-(pyrene-1-yl)-butyric acid methylester

To 40 ml of methanol, 20 ml (260 mmol) of thionyl chloride were dropwiseadded at 0° C. Then, the solution was stirred at room temperature for 1h. After addition of 5.50 g (19 mmol) of 4-(pyrene-1-yl)-butyric acid,the preparation was stirred over night at room temperature. The reactionwas neutralized with saturated NaHCO₃ solution and extracted with aceticacid ethylester. An aliquot was taken from the organic phase foranalysis. The remainder of the crude product was used further to prepare4-(pyrene-1-yl)-butane-1-ol.

M (C₂₁H₁₈O₂)=302 g/mol

EI-MS: m/z (%)=303 (13) [MH⁺], 302 (57) [M], 228 (15), 216 (22), 215(100), 213 (12), 108 (13). HR-EI-MS (C₂₁H₁₈O₂): calc.: 302.1307; found:302.1306. ¹H-NMR (CDCl₃): δ (ppm)=8.25-7.70 (9H, m, CH_(arom)), 3.63(3H, s, CH₃), 3.27 (2H, t, CH₂C₁₆H₉, ³J_(HH)=7.60 Hz), 2.36 (2H, t,CH₂CO, ³J_(HH)=7.20 Hz), 2.11 (2H, m, CH₂, ³J_(HH)=7.20 Hz, ³J_(HH)=7.60Hz). ¹³C-NMR (CDCL₃): δ (ppm)=173.73 (1 CO), 135.51-123.15 (9 CH_(arom),7 C_(arom)), 51.38 (1 CH₃), 33.52-26.64 (1 CH₂CO, 2 CH₂). IR (KBr):{tilde over (ν)} (cm⁻¹)=3033w, 2949w, 2876w, 1734s, 1429m, 1209m, 1161m,842s.

4-(pyrene-1-yl)-butane-1-ol

The crude product from the previous step was dissolved in 30 ml of THFat 0° C., with 3 g (79 mmol) of LiAlH₄ being added thereto. The reactionwas stirred over night at room temperature. Then, excessive reducingagent was hydrolysed. Column chromatography yielded 4.6 g (92%, in 2steps) of the desired compound.

M (C₂₀H₁₈O)=274 g/mol

EI-MS: m/z (%)=275 (10) [MH⁺], 274 (53) [M], 216 (17) 215 (100).HR-EI-MS (C₂₀H₁₈O): calc.:274.1358; found: 274.1357. ¹H-NMR (CDCl₃): δ(ppm)=8.23-7.79 (9H, m, CH_(arom)), 3.64 (2H, t, CH₂O, ³J_(HH)=6.39 Hz),3.31 (2H, t, CH₂C₁₆H₉, ³J_(HH)=7.60 Hz), 187 (2H, m, CH₂, ³J_(HH)=7.70Hz, ³J_(HH)=7.60 Hz), 1.69 (2H, m, CH₂, ³J_(HH)=7.70 Hz, ³J_(HH)=6.39Hz). ¹³C-NMR (CDCl₃): δ (ppm)=136.65-123.35 (9 CH_(arom), 7 C_(arom)),62.77 (1 CH₂O), 33.17-27.93 (3 CH₂). IR (KBr): {tilde over (ν)}(cm⁻¹)=3423s, br, 3038m, 2927m, 2859m, 1181w, 1030w, 982w, 841s.

1-[4-(10-bromo-decyloxy)-butyl]-pyrene

2 g (7.3 mmol) of 4-(pyrene-1-yl)-butane-1-ol were heated at 100° C. for1 hour in 50 ml of toluene containing 1 g (9 mmol) ofpotassium-tert-butylate. Then, 2.22 g (7.4 mmol) of 1,10-dibromodecanewere added and heated for 16 further hours. Column chromatographyyielded 2.82 g (79%) of a light yellow solid.

M (C₃₀H₃₇O⁷⁸Br)=492 g/mol

EI-MS: m/z (%)=495 (10) [MH⁺], 494 (31) [M], 493 (10) [MH⁺], 492 (33)[M], 412 (13), 228 (22), 215 (100), 55 (18), 41 (13). HR-EI-MS(C₃₀H₃₇O⁷⁸Br): calc.: 492.2028; found: 492.2028. ¹H-NMR (CDCl₃): δ(ppm)=8.30-7.85 (9H, m, CH_(arom)), 3.49-3.45 (2H, t, CH₂Br,³J_(HH)=6.44 Hz), 3.41-3.34 (6H, m, CH₂C₁₆H₉, 2 CH₂O, ³J_(HH)=7.08 Hz,³J_(HH)=6.81 Hz), 1.96-1.90 (2H, m, CH₂, ³J_(HH)=7.08 Hz, ³J_(HH)=6.44Hz), 1.83-1.73 (4H, m, 2 CH₂, ³J_(HH)=6.81 Hz), 1.55-1.53 (2H, m, CH₂),1.25 (12H, m, 6 CH₂). ¹³C-NMR (CDCl₃): δ (ppm)=136.72-123.30 (9CH_(arom), 7 C_(arom)), 70.93-70.55 (2 CH₂O), 33.94-26.15 (1 CH₂Br, 11CH₂). IR (KBr): {tilde over (ν)} (cm⁻¹)=3037w, 2924s, 2851s, 1499m,1448m, 1429w, 1369w, 1243w, 1184w, 1117s, 1063w, 965w, 849m, 841s, 710s.

2,2-dimethyl-4-[10-O-(4-(pyrene-1-yl)-butoxy)decyl)-methyl]-[1,3]-dioxolane

1.85 g (14 mmol) of 2,2-Dimethyl-4-hydroxymethyl-1,3-dioxolan wereheated with reflux for 1 hour in 80 ml of toluene containing 1.6 g (14mmol) of potassium-tert-butylate. After cooling to room temperature,2.118 g (4.3 mmol) of 1-[4-(10-bromo-decyloxy)-butyl]-pyrene were addedand heated for 18 further hours. Column chromatography yielded 1.174 g(50%) of a light yellow solid.,

M (C₃₆H₄₈O₄)=544 g/mol

EI-MS: m/z (%)=545 (22) [MH⁺], 544 (62) [M], 257 (22), 228 (24), 216(22), 215 (100), 101 (18), 55 (11), 43 (14). HR-EI-MS (C₃₆H₄₈O₄): calc.:544.3553; found: 544.3553. ¹H-NMR (CDCl₃): δ (ppm)=8.29-7.83 (9H, m,CH_(arom)), 4.26-4.21 (2H, m, CHCH₂O), 4.07-4.00 (1H, m, CHO), 3.75-3.31(10H, m, 4 OCH₂, CH₂C₁₆H₉), 1.95-1.86 (2H, m, CH₂), 1.78-1.72 (2H, m,CH₂), 1.55-1.53 (4H, m, CH₂), 1.41 und 1.35 (6H, s, 2 CH₃), 1.26 (12H,m, 6 CH₂). ¹³C-NMR (CDCl₃): δ (ppm)=136.92-123.48 (9 CH_(arom), 7C_(arom)), 109.33 (1 C(CH₃)₂), 74.77 (1 CHO), 71.85-70.64 (4 CH₂O),66.94 (1 CH₂O), 33.33-25.89 (11 CH₂), 26.77 und 25.43 (2 CH₃). IR (KBr):{tilde over (ν)} (cm⁻¹)=3059m, 2952s, 2890m, 2866s, 1524w, 1467m, 1369w,1289m, 11121m, 1058m, 837w, 684s.

1-O-{10-[4-(pyrene-1-yl)-butoxy]-decyl}-rac-glycerol

1 ml trifluoroacetic acid was added to 0.183 g (0.336 mmol) of2,2-dimethyl-4-[10-O-(4-(pyrene-1-yl)-butoxy)-decyl)-methyl]-[1,3]-dioxolanein 10 ml of toluene at 0° C. and stirred for one hour. Afterneutralization with an aqueous solution of NaHCO₃, column chromatographyyielded 0.160 g (94%) of a light yellow solid.

M (C₃₃H₄₄O₄)=504 g/mol

EI-MS: m/z (%)=505 (32) [MH⁺], 504 (100) [M], 257 (17), 228 (21), 216(19), 215 (89), 55 (14). HR-EI-MS (C₃₃H₄₄O₄): calc.: 504.3240; found:504.3238. ¹H-NMR (CDCl₃); δ (ppm)=8.29-7.83 (9H, m, CH_(arom)),3.87-3.59 (3H, m, CHOH, CH₂OH), 3.47-3.31 (10H, m, 4 OCH₂, CH₂C₁₆H₉),1.94-1.89 (2H, m, CH₂), 1.77-1.72 (2H, m, CH₂), 1.54-1.51 (4H, m, 2CH₂), 1.24 (12H, m, 6 CH₂). ¹³C-NMR (CDCl₃): δ (ppm)=136.94-123.48 (9CH_(arom), 7 C_(arom)), 72.03-70.65 (4 CH₂O), 70.50 (1 CHOH), 63.81 (1CH₂OH), 33.31-25.97 (11 CH₂). IR (KBr): {tilde over (ν)}(cm⁻¹)=3419s,br, 3058w, 2932m, 2851m, 1437m, 1433m, 1265m, 1205m, 1186m,1138m, 1112m, 1048m, 843m.

1-O-{10-[4-(pyrene-1-yl)-butoxy]-decyl}-3-O-triphenylmethyl-rac-glycerol

4.215 g (8.4 mmol) of1-O-{10-[4-(pyrene-1-yl)-butoxy]-decyl}-rac-glycerol were prepared in 50ml of acetonitrile, to which 4.09 g (10 mmol) of1-(triphenylmethyl)pyridinium-tetrafluoroborate were added in 20 ml ofacetonitrile. The solution was stirred for 18 hours, concentrated andtaken up by chloroform. The solid was filtered off. Columnchromatography of the filtrate yielded 3.697 g (59%) of the desiredcompound as a light yellow solid.

M (C₅₂H₅₈O₄)=746 g/mol

FAB-MS (FAB⁺, NBA): m/z=746.4 [M], 504.3, 244.1, 215.1. HR-FAB(C₅₂H₅₈O₄) calc.: 746.4335 found: 746.4375. HR-FAB (C₅₂H₅₈O₄Na): calc.:769.4233; found: 769.4304. ¹H-NMR (CDCl₃): δ (ppm)=8.31-7.85 (9H, m,CH_(arom)), 7.45-7.19 (15H, m, CH_(arom)), 3.95 (1H, m, CH), 3.55-3.33(10H, m, 4 OCH₂, CH₂C₁₆H₉), 3.24-3.18 (2H, m, CH₂OC(C₆H₅)₃), 1.97-1.87(2H, m, CH₂), 1.81-1.73 (2H, m, CH₂), 1.55-1.51 (4H, m, 2 CH₂), 1.25(12H, m, 6 CH₂). ¹³C-NMR (CDCl₃): δ (ppm)=146.90-123.51 (24 CH_(arom),10 C_(arom)), 86.65 (1 C(C₆H₅)₃), 72.95-70.67 (4 CH₂O), 69.87 (1 CH),64.65 (1 CH₂OC(C₆H₅)₃), 33.35-26.97 (11 CH₂). IR (KBr): {tilde over (ν)}(cm⁻¹)=3429m, 3056w, 2927s, 2855s, 1602w, 1490m, 1448m, 1319w, 1213m,1113m, 1077m, 1033m, 899w, 846m, 746m, 706s.

1-O-{10-[4-(pyrene-1-yl)-butoxy]-decyl}-2-O-benzoyl-3-O-triphenylmethyl-rac-glycerol

To a solution of 2.578 g (3.455 mmol) of1-O-{10-[4-(pyrene-1-yl)-butoxy]-decyl}-3-O-triphenylmethyl-rac-glycerol and 1.5 ml (18.66 mmol) of pyridine in 50ml of CH₂Cl₂, 540 mg (3.857 mmol) of benzoylchloride were added dropwiseat 0° C. Benzoylchlorid and stirred at room temperature for 1 hour.After column chromatography, 2.8 g (95%) of the product were isolated.

M (C₅₉H₆₂O₅)=850 g/mol

FAB-MS (FAB⁺, NBA): m/z=873.4 [M+Na], 850.4 [M], 608.3, 591.3, 391.3,307.1, 243.1, 215.1. ¹H-NMR (CDCl₃): δ (ppm)=8.30-7.86 (9H, m,CH_(arom)), 7.45-7.21 (20H, m, CH_(arom)), 5.46-5.41 (1H, m, CH),3.79-3.34 (12H, m, 4 CH₂O, CH₂C₁₆H₉, CH₂OC(C₆H₅)₃), 1.97-1.89 (2H, m,CH₂),1.80-1.72 (2H, m, CH₂), 1.55-1.51 (4H, m, CH₂), 1.25 (12H, m, CH₂).¹³C-NMR (CDCl₃): δ(ppm)=166.00 (1 CO), 146.88-123.51 (29 CH_(arom), 11C_(arom)), 86.47 (1 C(C₆H₅)₃), 72.65 (1 CH), 71.59-69.47 (4 CH₂O), 62.86(1 CH₂OC(C₆H₅)₃), 33.35-26.01 (11 CH₂). IR (KBr): {tilde over (ν)}(cm⁻¹)=3057w, 2927s, 2854m, 1718s, 1448m, 1384m, 1272m, 1212w, 1177w,1158w, 1112m, 1032w, 899w, 846m, 763m, 703m.

1-O-{10-[4-(pyrene-1-yl)-butoxy]-decyl}-2-O-benzoyl-rac-glycerol

0.55 g (0.647 mmol) of1-O-{10-[4-(pyrene-1-yl)-butoxy]-decyl}-2-O-benzoyl-3-O-tri-phenylmethyl-rac-glycerol were dissolved in 20 ml CH₂Cl₂, and 0.5ml BF₃ (13% in MeOH) was added dropwise thereto at −10° C. The reactionwas stopped with 5 ml of iced water after one hour. Columnchromatography yielded 280 mg (71%) of the compound.

M (C₄₀H₄₈O₅)=608 g/mol

FAB-MS (FAB⁺, NBA): m/z=608.3 [M], 591.3, 469.3, 329.1, 257.2, 215.1.HR-FAB (C₄₀H₄₈O₅): calc.: 608.3502; found: 608.3519. HR-FAB(C₄₀H₄₈O₅Na): calc.: 631.3399; found: 631.3424. ¹H-NMR (CDCl₃): δ(ppm)=8.29-7.84 (9H, m, CH_(arom)), 7.62-7.34 (5H, m, CH_(arom)),5.27-5.22 (1H, m, CH), 3.99-3.34 (12H, m, 4 CH₂O, CH₂C₁₆H₉, CH₂OH),1.97-1.88 (2H, m, CH₂), 1.80-1.72 (2H, m, CH₂), 1.57-1.51 (4H, m, 2CH₂), 1.25 (12H, m, 6 CH₂). ¹³C-NMR (CDCl₃): δ (ppm)=166.38 (1 CO),136.94-123.51 (14 CH_(arom), 8 C_(arom)), 73.81 (1 CH), 71.94-68.95 (4CH₂O), 62.87 (1 CH₂OH), 33.80-26.19 (11 CH₂). IR (KBr): {tilde over (ν)}(cm⁻¹)=3431m, 3038m, 2930s, 2859m, 1718s, 1603m, 1585m, 1453m, 1435m,1317m, 1274s, 1179m, 1113m, 1027m, 936m, 846m, 709s.

1-O-{-10-[4-(pyrene-1-yl)-butoxy]-decyl}-2-O-benzoyl-rac-glycero-3-phosphocholine

0.55 g (2.3 mmol) of 2-bromoethyl-dichlorophosphate were added to 15 mlof CH₂Cl₂ at 0° C., and a mixture of 1.6 ml of pyridine and 4 ml ofCH₂Cl₂ was added dropwise thereto. Then, 210 mg (3.45 mmol) of1-O-{10-[4-(pyrene-1-yl)-butoxy]-decyl}-2-O-benzoyl-rac-glycerol weredissolved in 2 ml of CH₂Cl₂ and added dropwise to the phosphorylationmixture at room temperature. The reaction was heated with reflux for 2hours and stirred over night at room temperature. After hydrolysis with5 ml of iced water (subsequent stirring for 1 hour), the reaction wasshaken with CHCl₃/MeOH (3:2). The organic phase was dried over Na₂SO₄,rotated and dissolved in 15 ml of CHCl₃, 10 ml of acetonitrile and 5 mlof iso-propanol. Then, 1.5 g of a trimethylamine solution (33% inethanol) were added. The reaction flask was tigthly sealed and stirredover night at 50° C. The reaction solution was rotated, taken up by 5 mlof CHCl₃ and shaken twice with each of 4 ml of formic acid/Me OH (5:7),4 ml of sodium acetate (0.1 M)/MeOH (5:7) and 3 ml of sodium chloride (1M)/MeOH (5:7). Drying over NaSO₄ and column chromatography of theorganic phase yielded 99 mg (38%) of the desired compound.

M (C₄₅H₆₀N₂O₈ ³¹P)=773 g/mol

FAB-MS (FAB⁺, NBA): m/z=796.3 [M+Na], 774.4 [MH⁺], 601.5, 569.4, 531.4,413.3, 215.1. HR -FAB (C₄₅H₆₁O₈NP): calc.: 774.4145; found: 774.4135.HR-FAB (C₄₅H₆₀O₈NPNa): calc.: 796.3954; found: 796.3951. ¹H-NMR (CD₃OD):δ (ppm)=8.34-7.89 (9H, m, CH_(arom)), 7.73-7.45 (5H, m, CH_(arom)),5.41-5.37 (1CH, m, CH), 4.20-4.17 (2H, m, CH₂N), 4.05-3.35 (14H, m, 4OCH₂, CH₂OP, POCH₂, CH₂C₁₆H₉), 3.16 (9H, s, 3 CH₃), 1.96-1.90 (2H, m,CH₂), 1.81-1.72 (2H, m, CH₂), 1.52-1.48 (4H, m, 2 CH₂), 1.28 (12H, m, 6CH₂). ¹³C-NMR (CD₃OD): δ (ppm)=167.78 (1 CO), 138.30-124.60 (14CH_(arom), 8 C_(arom)), 74.27 (1 CH), 72.81-70.48 (4 CH₂O), 68.87,66.90, 60.95 (1 CH₂N, 1 CH₂OP, 1 POCH₂), 54.90 (3 CH₃), 34.38-27.29 (11CH₂).

1-O-{(10-[4-(pyrene-1-yl)-butoxy]-decyl}-rac-glycero-3-phosphocholine

90 mg (0.12 mmol) of1-O-{10-[4-(pyrene-1-yl)-butoxy]-decyl}-2-O-benzoyl-rac-glycero-3-phosphocholinewere dissolved in 3 ml of MeOH, and 1.5 ml of a 0.1 M Bu₄NOH solutionwas added thereto in MeOH (1.25 eq). The reaction solution wasconcentrated after 2 hours. Column chromatography yielded 71 mg (90%) ofthe lyso-compound.

M (C₃₈H₅₆NO₇ ³¹P)=669 g/mol

FAB-MS (FAB⁺, NBA): m/z=692.3 [M+Na], 670.4 [MH⁺], 669.4 [M], 584.3,242.3, 289.1, 215.1. HR-FAB (C₃₈H₅₇NO₇ ³¹P): calc.: 670.3873; found:670.3881. HR-FAB (C₃₈H₅₆NO₇ ³¹PNa): calc.: 692.3692; found: 692.3693.¹H-NMR (CD₃OD): δ (ppm)=8.23-7.88 (9H, m, CH_(arom)), 4.43-4.38 (2H, m,CH₂N), 4.02-3.36 (15H, m, 4 OCH₂, OCH, CH₂OP, POCH₂, CH₂C₁₆H₉), 3.22(9H, s, 3 CH₃), 1.96-1.87 (2H, m, CH₂), 1.77-1.66 (2H, m, CH₂),1.55-1.47 (4H, m, 2 CH₂), 1.27 (12H, m, 6 CH₂). ¹³C-NMR (CDCl₃): δ(ppm)=170.46 (1 CO), 138.16-124.48 (9 CH_(arom), 7 C_(arom)),72.90-71.47 (4 CH₂O), 71.03 (1 CH), 67.31, 66.63, 59.33 (1 CH₂OP, 1CH₂N, 1 POCH₂), 54.78 (3 CH₃), 33.36-24.59 (10 CH₂). IR (KBr): {tildeover (ν)} (cm⁻¹)=3418s, 3039m, 2852m, 1635m, 1490m, 1467m, 1375w, 1230m,1111m, 1087m, 1051m, 970m, 928w, 850m.

1O-{10-[4-pyrene-1yl)-butoxy]decyl}-2-(6-(2,4dinitrophenyl)-aminohexoyl)-rac-glycero-3-phosphocholine

0.326 g (1.1 mmol) of 6-(2,4-dinitrophenylamino)-hexane acid, 136 mg(0.66 mmol) of DCC and 408 mg (3.2 mmol) of DMAP were added to 0.150 g(0.22 mmol) of 1-O-{10-[4-(pyrene-1-yl)-butoxyl]-decyl}-rac-glycero-3-phosphocholine in 10 ml ofdichloromethane and stirred for 40 hours at room temperature. To theresulting suspension 5 ml of water were then added and filtered overkieselguhr. After separation of the phases and chromatography, 56 mg(26%) of the product were isolated.

M (C₅₀H₆₉N₄O₁₂ ³¹P)=948 g/mol

FAB-MS (FAB⁺, NBA): m/z=971.4 [M+Na], 949.4 [MH⁺], 884.4, 862.4, 721.4,243.2, 215. 1. HR-FAB (C₅₀H₇₀O₁₂N₄P): calc.: 949.4725; found: 949.4755.HR-FAB (C₅₀H₆₉O₁₂N₄PNa): calc.: 971.4545; found: 971.4547. ¹H-NMR(CDCl₃): δ (ppm)=9.00-6.67 (12H, m, CH_(arom)), 5.13-5.10 (1H, m, CH),4.34-4.28 (2H, m, CH₂N), 4.08-3.23 (16H, m, 4 OCH₂, POCH₂, CH₂OP, CH₂N,CH₂Cl₁₆H₉), 3.18 (9H, s, 3 CH₃), 2.38-2.31 (2H, m, CH₂CO), 1.95-1.86(4H, m, 2, CH₂), 1.80-1.72 (2H, m, CH₂), 1.56-1.41 (8H, m, 4 CH₂), 1.25(12H, m, 6 CH₂). ¹³C-NMR (CDCl₃): δ (ppm)=170.46 (1 CO), 148.66-113.98(12 CH_(arom), 10 C_(arom)), 73.86 (1 CH), 72.81-69.12 (4 CH₂O), 67.31,66.63, 60.30 (1 CH₂OP, 1 CH₂N, 1 POCH₂), 54.78 (3 CH₃), 43.27 (1 CH₂N),33.36-24.59 (1 CH₂CO, 14 CH₂) IR (KBr): {tilde over (ν)} (cm⁻¹)=3365m,3107w, 3042w, 2928m, 2853m, 1729m, 1623s, 1589m, 1525m, 1500w, 1475w,1425m, 1369w, 1337s, 1312m, 1280m, 1245m, 1187m, 1144m, 1094m, 1060w,1032w, 923w, 832w, 745w.

EXAMPLE 2 Determination of the Activity of PAF-AH Using1-O-{10-[4-(pyrene-1-yl)-butoxy]-decyl}-2-(6-(2,4-dinitrophenyl)-aminohexoyl)-rac-glycero-3-phosphocholineAccording to the Invention

For the examinations, 10 mM of DMSO stock solution of the compoundaccording to the invention of Example 1 were prepared and thenrespectively diluted with the buffer of use (0.1 M TRIS HCl, 0.1%Tween20, pH 7.4) at the desired concentrations for the respective tests.

Measurements in a Quartz Cuvette

Use is made of quartz cuvettes (1 cm×1 cm) of Helma (Müllheim/Baden,Germany) with a volume of 1 ml.

Settings in the Cary Eclipse software of Varian (Darmstadt, Germany):

-   -   In the kinetics module: Ex. Wavelength (nm)=344.00; Em.        Wavelength (nm)=377.00; Ex. Slit (nm)=5; Em. Slit (nm)=5;        Averaging Time (s)=0.1000; Dwell time (s)=0.1000; Cycle time        (min)=0.2500; Excitation filter=Auto; Emission filter=Open;        Multicell holder=Multicell; PMT voltage (V)=Medium; Temperature        control.

The examinations were carried out using a four-way Peltier cuvetteholder at 37° C.

Measurements in a Microtiter Plate

Use was made of white 96-well titer plates of Perkin Elmer (Überlingen,Germany) and black 96-well FIA plates of Greiner (Frikenhausen,Germany).

Settings in the Cary Eclipse software of Varian (Darmstadt, Germany):

-   -   In the kinetics module: Ex. Wavelength (nm)=344.00; Em.        Wavelength (nm)=377.00; Ex. Slit (nm)=5; Em. Slit (nm)=5;        Averaging Time (s)=0.1000; Cycle time (min)=0.0000; Excitation        filter=Auto; Emission filter=Open; PMT voltage (V)=Medium;        Wellplate=ON; Read position=Well centre.        Preparation of PAF-AH        Preparation and Purity Examination of Plasma and Human LDL

Buffer used: PBS buffer (150 mM NaCl, 40 mM phosphate, 1 mM EDTA,pH=7.4). The buffer was saturated with argon for 15 min.

Preparation of Plasma

100 ml of freshly collected, heparinized blood was centrifuged for 10min in 2×50 ml tubes at room temperature, using a Hermle centrifuge at3,000 rpm. 20 ml were taken from each supernatant, pooled, 400 μl of 0.1M EDTA solution (pH=7.45) added thereto, and stratified with argon.

Preparation of LDL

12.2112 g of KBr p.a. was added to 32 ml of the plasma sample andstratified with argon. The KBr was dissolved by slow oscillation. 2 mlof the PBS solution were prepared in 6×4 ml centrifuge tubes of Beckman(Munich, Germany) and the plasma sample, with KBr added thereto, wasadded to form a layer. This was then centrifuged at 4° C. and at 37,000rpm in a vacuum for 2 hours. After this time, the centrifuge tubes werecarefully removed from the rotor.

The desired LDL is distinguishable as a yellow band, at a distance ofabout ⅓ from the bottom of the centrifuge tube. At first, the upperwhite layer was removed and discarded. The yellow layer is about 1 mmthick. Said layer was removed using a blunt, bent needle. The LDLcontained in all 6 centrifuge tubes was pooled and stratified withargon. In order to remove the KBr, 300 μl of the thus obtained solutionwere chromatographed by means of a Sephadex-G25 minicolumn using PBSbuffer.

Protein Measurement

Solution A: 1% Na₂BCA, 2% Na₂CO₃ H₂O, 0.16% Na₂tartrate, 0.4% NaOH,0.95% NaHCO₃, pH=11.25

Solution B: 4% CuSO₄ 5 H₂O

BSA was used as the standard; all measurements were carried out asdouble measurements. The total volume of one preparation was 300μl/well, and usually 2.5 μl of the sample to be determined (afterdilution, as the case may be) were employed. This was filled up to 75 μlwith water, and 225 μl of a 50:1 mixture of solution A and solution Bwere added thereto. The incubation time was 2 hours at room temperature.The samples were then photometrically measured at 562 nm.

Examination of the Purity of the Isolated LDL

The examination was carried out using submaritime mini gelelectrophoresis.

-   -   Gel composition: 20 ml of barbital buffer (50 mM, pH=8.6), 0.3%        Separide™ (60 mg)    -   Preparation of the isolated LDL: 10 μl (0.5-1 μg) of columned        LDL, 0.5 μl of 9-diethylamino-5H[a]phenoxazene-5-one (Nile Red)        (0.4 mg/ml), 10 μl of application buffer (TBE buffer, 30%        glycerol, 1% SDS, spatula tip of Bromphenol Blue)    -   TBE buffer (89 mM TRIS HCl, 89 mM B(OH)₃, 2 mM EDTA, pH=8.5)    -   Duration of electrophoresis: 1 h (at a constant U of 56 V) at        room temperature    -   Fixing solution: 25% ^(i)PrOH, 10% acetic acid, 65% water    -   Staining solution: 50% MeOH, 10% acetic acid, 40% water, 0.05%        Coomassie Brilliant Blue R 250    -   Destaining solution: 5% MeOH, 7% acetic acid, 88% water        Isolation of PAF-AH from LDL

Buffer A: 25 mM TRIS HCl, 0.1% Tween20, 2 mM EDTA, pH 7.6

Buffer B: 25 mM TRIS HCl, 0.1% Tween20, 2 mM EDTA, 50-200 mM NaCl, pH7.6 (linear gradient)

All work was carried out at 4° C.

1 ml (2 mg/ml) LDL was pipetted onto a pre-equilibrated (Buffer A) DEAESepharose® FF chromatography column of Amersham Pharmacia biotech(Freiburg, Germany) and washed with 6 times the column filling volume togive the protein-free eluent. Next, the protein was eluated with bufferB. The highest PAF-AH activity was obtained with fractions of 100-150 mMNaCl. These were pooled and concentrated by ultrafiltration (AmiconYM30). The upgraded fractions were then pipetted onto a pre-equilibrated(Buffer A) Blue Sepharose® 6FF chromatography column of AmershamPharmacia biotech (Freiburg, Germany) and washed with 6 times the columnfilling volume to give the protein-free eluent. Next, the protein waseluated with buffer A+0.5 M NaCl. The fractions having the highestPAF-AH activity were pooled and concentrated by ultrafiltration (AmiconYM30).

The content of PAF-AH was determined by protein determination. Puritywas examined by analogy with the SDS electrophoresis of LDL. Theresulting PAF-AH was sufficiently pure for activity examinations.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the time course of the increase in fluorescence by theeffect of PAF-AH on the1-O-{10-[4-(pyrene-1-yl)-butoxy]-decyl}-2-(6-(2,4-dinitrophenyl)-aminohexoyl)-rac-glycero-3-phos-phocholinecompound according to the invention. The measurement was carried outunder the conditions described above. As is apparent from FIG. 1, anincrease in fluorescence occurs in the presence of PAF-AH. No increasein the intensity of fluorescence was observed without PAF-AH (control).

FIG. 2 shows the connection between different amounts of PAF-AH ordifferent dilutions of human sample material and the increase in theintensity of fluorescence (test conditions as above). It was found thata marked increase in the intensity of fluorescence can be observed athigher PAF-AH concentrations.

1. A compound of formula (1)

wherein L₁ comprises an ether (R¹—OR²)_(m), wherein R¹ and R² areindependently selected from a hydrocarbon having 1 to 12 carbon atoms,with m being an integer from 1 to 4, or from a hydrocarbon R having 1 to20 carbon atoms, and wherein a carbon atom of L₁ is bonded to an sn-1positioned O atom; F comprises pyrene as a fluorophore; Q is a quencher,and L₂ is C(O)-L₁ or C(O)-L₁-NH, wherein L₁ is as defined above.
 2. Thecompound of claim 1, wherein the quencher comprises a 2,4-dinitrophenylresidue.
 3. The compound of claim 1, wherein R¹ and R² are independentlyselected as (CH₂)_(n) and a is an integer from 2 to
 12. 4. The compoundof claim 1, wherein L₁ is (CH₂)₄—O—(CH₂)₁₀.
 5. The compound of claim 1,wherein R is (CH₂)_(o) and o is an integer from 1 to
 20. 6. The compoundof claim 1, wherein L₂ is C(O)—(CH₂)_(p) or C(O)—(CH₂)_(p)—NH and p isan integer from 1 to
 20. 7. The compound of claim 6, wherein L₂ isC(O)—(CH₂)₅—NH.
 8. The compound of claim 1, wherein said compound hasthe structure of:


9. The compound of claim 1, wherein F is a substituted pyrene.
 10. Amethod for detecting activity of phospholipase A₂, present in asolution, comprising reacting the solution with a compound having theformula

wherein L₁ comprises an ether (R¹—OR²)_(m), wherein R¹ and R² areindependently selected from a hydrocarbon having 1 to 12 carbon atoms,with m being an integer from 1 from 4, or from a hydrocarbon R having 1to 20 carbon atoms, and wherein a carbon atom of L₁ is bonded to an sn-1positioned O atom; F comprises nyrene as a fluorophore; O is a quencher,L₂ is C(O)—L₁ or C(O)-L₁-NH, wherein L₁ is as defined above; anddetecting activity of phospholipase A₂ by measuring the level offluorescence intensity of the fluorophore in the solution.
 11. Themethod of claim 10, wherein the quencher comprises a 2,4-dinitrophenylresidue.
 12. The method of claim 10, wherein R¹ and R² are independentlyselected as (CH₂)_(n) and n is an integer from 2 to
 12. 13. The methodof claim 10, wherein R is (CH₂)_(o) and o is an integer from 1 to 20.14. The method of claim 10, wherein L₂ is C(O)—(CH₂)_(p) orC(O)—(CH₂)_(p)—NH and p is an integer from 1 to
 20. 15. The method ofclaim 10, wherein L₂ is C(O)—(CH₂)₅—NH.
 16. The method of claim 10,wherein said compound has the structure of:


17. The method of claim 10, wherein the phospholipase A₂ isPlatelet-Activating Factor Acetylhydrolase.